1. Volume 10, Issue 1, Pages 44-53 (July 2011)
Lymphotoxin Controls the IL-22 Protection Pathway in Gut Innate Lymphoid Cells during Mucosal Pathogen Challenge 
Alexei V. Tumanov, Ekaterina P. Koroleva, Xiaohuan Guo, Yugang Wang, Andrei Kruglov, Sergei Nedospasov, Yang-Xin Fu 
Cell Host & Microbe 
Volume 10, Issue 1, Pages (July 2011)
DOI: /j.chom
Copyright © 2011 Elsevier Inc. Terms and Conditions

2. Figure 1 LTβR Signaling Controls the Innate IL-22 Pathway in the Gut
(A) IL-22 levels in the colon of naive and infected WT and Ltbr−/− mice. LTbr−/− and WT mice (n = 5/group/experiment) were orally inoculated with C. rodentium. mRNA expression of IL-22 and IL-22- dependent RegIIIγ and RegIIIβ antimicrobial proteins in the colon were measured by real-time PCR on day five postinfection. Naive mice were used as control.
(B) WT mice were treated with 150 μg of LTβR-Ig, or control human Ig (hIg) at day zero and three postinfection. Expression of IL-22 and RegIIIγ and RegIIIβ antimicrobial proteins were measured in the colon by real-time PCR at day five postinfection.
(C) Rag1−/− mice were treated intraperitoneally with 150 μg of LTβR-Ig at day zero and three postinfection, and expression of IL-22, and RegIIIγ and RegIIIβ antimicrobial proteins was measured by real-time PCR at day five postinfection. (All panels) Data represent means ± SEM. n = 5, ∗p < 0.05, ∗∗p < Data represent one of three independent experiments with similar results. Real-time PCR data were normalized to hprt expression.
Cell Host & Microbe  , 44-53DOI: ( /j.chom )
Copyright © 2011 Elsevier Inc. Terms and Conditions

3. Figure 2
IL-22 Is an Essential Protection Pathway Downstream of LTβR Signaling
(A–C) Stimulation of LTβR signaling promotes the IL-22 protection pathway. Ltb−/− mice were injected intraperitoneally with 75 μg of agonistic anti-LTβR antibody or control rat Ig (rIg) at day zero and three postinfection with C. rodentium. mRNA expression of IL-22 (A) and RegIIIγ (B) were measured in the colon by real-time PCR at day four postinfection. Bacterial titers in liver are shown at day four postinfection (C). n = 4, ∗∗p < Data represent means ± SEM.
(D–G) Hydrodynamic injection of IL-22-expressing plasmid rescues Ltbr−/− mice from lethal C. rodentium infection. IL-22-expressing plasmid or control vector was intravenously injected to WT or Ltbr−/− mice 6 hr after C. rodentium infection. n = 10–13/group. Survival (D) and body weight change (E) are shown. ∗∗∗p < between IL-22-treated Ltbr−/− and control untreated Ltbr−/− mice by Mantel-Cox log rank test. Data combined from two experiments with similar results. Representative hematoxylin and eosin staining of colons is shown at day nine postinfection (F). Red boxes in top panels are shown at higher magnification in lower panels. Bars: 1 mm for top panels and 200 μm for lower panels. Colitis histopathology score (G). n = 5, ∗p < Data represent means ± SEM.
Cell Host & Microbe  , 44-53DOI: ( /j.chom )
Copyright © 2011 Elsevier Inc. Terms and Conditions

4. Figure 3
IL-22 Is Produced in Lymphoid Follicles by Innate RORγt+ Cells
(A) IL-22 is predominantly expressed in the lamina propria. Colon lamina propria (LP) and intraepithelial lymphocyte (IEL) cell populations from WT mice were purified at day five postinfection. IL-22 expression was measured by real-time PCR. Data were normalized to hprt expression. Data represent means ± SEM. n = 5 mice, ∗∗p < 0.01.
(B) LTi cells are predominant IL-22-producing cells in the lamina propria. Colon LP innate cell populations from RORγt-GFP+/−/Ltbr+/+ and RORγt-GFP+/−/LTbr−/− mice at day five postinfection were purified by flow cytometry. IL-22 expression was measured by real-time PCR. Sorted cell populations are indicated as LTi: CD45+CD3−GFP+NK1.1−NKp46−; NKp46: CD45+CD3−GFP+NKp46+; NK: CD45+CD3−GFP−NK1.1+; and Th17: CD45+CD3+GFP+NK1.1−NKp46−. Data were combined from two experiments and represent means ± SEM. n = 4, ∗∗p < IL-22 data were normalized to hprt expression.
(C) IL-22-expressing cells are predominantly located in lymphoid follicles. WT mice were orally infected with C. rodentium, and colon sections at day five postinfection were stained with indicated antibodies. Nuclei were stained with DAPI. Bars: 20 μm.
(D) IL-22-producing cells interact with DC in lymphoid follicles. Colon sections of mice at day five postinfection were stained with indicated antibodies and analyzed by confocal microscopy. Nuclei were stained with DAPI. Arrows indicate contact of DC (green) with IL-22-producing cells (red) on top panel and RORγt+CD4− cells on bottom panel. Bars: 10 μm.
(E) IL-22 is expressed predominantly in isolated lymphoid follicles. WT mice were orally infected with C. rodentium, and colon tissue was collected at day five postinfection. Isolated lymphoid follicles (ILF+) and surrounding tissue (ILF−) were microdissected under stereo microscope, and IL-22 expression was measured by real-time PCR. IL-22 data were normalized to hprt expression. ∗p < 0.05, n = 3 mice, means ± SEM.
(F) Coculture of LTi cells with DC promotes IL-22 production by LTi cells. Lamina propria LTi cells from naive RORγt-GFP+/− mice were cocultured in vitro for 18 hr with lamina propria DCs from WT mice at day five postinfection. After coculture, LTi and DC cells were separated by flow cytometry, and IL-22 was measured by real-time PCR. Data were normalized to β-actin. One of two independent experiments with similar results is shown.
Cell Host & Microbe  , 44-53DOI: ( /j.chom )
Copyright © 2011 Elsevier Inc. Terms and Conditions

5. Figure 4
LT Expression in RORγt+ Cells Is Essential for Control of IL-22 Production and Protection of Mice against C. rodentium Infection
(A and B) LT expression in RORγt+ cells controls the IL-22 protection pathway. WT and RORγt-Ltb−/− mice were orally infected with C. rodentium. Expression of IL-22 in colon and purified lamina propria cells (A) and in antimicrobial proteins RegIIIγ and RegIIIβ (B) was measured by real-time PCR at day five postinfection. Data represent means ± SEM. One out of three independent experiments with similar results is shown. n = 5 mice, ∗p < 0.05, ∗∗p < Expression data were normalized to hprt expression.
(C) IL-22 expression is sufficient to rescue RORγt-Ltb−/− mice from lethal C. rodentium infection. WT and RORγt-Ltb−/− mice were intravenously injected with IL-22-expressing plasmid or control vector at 6 hr after C. rodentium infection. n = 10/group. Data were combined from two experiments with similar results. Survival and body weight change are shown.
(D) Representative hematoxylin and eosin staining of the colon at day nine postinfection. Red boxes in top panel are shown at higher magnification in lower panel. Bars: 1 mm for top panel and 200 μm for lower panel.
(E) Colitis histopathology score for mice in panel (A). n = 5, ∗∗∗p < Data represent means ± SEM. One of two experiments with similar results is shown.
Cell Host & Microbe  , 44-53DOI: ( /j.chom )
Copyright © 2011 Elsevier Inc. Terms and Conditions

6. Figure 5 LTβR Signaling in DCs Is Required for IL-22 Production
(A and B) WT, CD11c-Ltbr−/−, and Ltbr−/− mice were orally infected with C. rodentium. Body weight change (A) and representative hematoxylin and eosin staining of colon at day 12 postinfection (B) are shown. Arrows indicate bacterial lesions. Bars: 50 μm (top panels) and 20 μm (lower panels).
(C–H) Bacterial titers were measured in feces (C) on day 14 and in liver (D) and spleen (E) on day 10 postinfection. Data represent IL-22 expression in the colon (F), IL-22 levels in colon supernatants (G), and expression of RegIIIγ mRNA in the colon (H), all at day five postinfection. Data represent means ± SEM. n = 5 mice, ∗p < 0.05, ∗∗p < One of three independent experiments with similar results is shown. Real-time PCR data were normalized to hprt expression.
(I) LTβR signaling in DCs regulates IL-23 production. CD11c+CD11b+ DCs from colon lamina propria of WT and LTbr−/− mice were purified by flow cytometry at day five postinfection. Expression of IL23p19, IL23p40, IL-1β, IL-6 cytokines was measured by real-time PCR. Data were combined from two experiments and represent means ± SEM. n = 4, ∗p < “NS”: not significant. RT-PCR data were normalized to hprt expression.
Cell Host & Microbe  , 44-53DOI: ( /j.chom )
Copyright © 2011 Elsevier Inc. Terms and Conditions

7. Figure 6
Proposed Model of How the IL-22 Pathway Defends against Mucosal Bacterial Pathogens
LT expression by RORγt+ ILCs is necessary for IL-22 production following invasion of mucosal bacterial pathogens. LT signaling by RORγt+ ILC promotes the development of lymphoid follicles in the gut. Lymphoid follicles provide the necessary microenvironment for interaction between innate RORγt+ cells and DC. Interplay between RORγt+ ILCs and DCs promotes IL-23 production by DCs that, in turn, activates IL-22 synthesis by RORγt+ cells as a positive feedback loop. IL-22 stimulates IL-22R on epithelial cells, which triggers production of antimicrobial proteins RegIIIγ and RegIIIβ to eliminate mucosal bacterial pathogens.
Cell Host & Microbe  , 44-53DOI: ( /j.chom )
Copyright © 2011 Elsevier Inc. Terms and Conditions